Novel ligands and methods for preparing same

ABSTRACT

A process is disclosed for modifying a parent ligand by attaching to the parent ligand a conjugation agent that is reactive with a moiety of a target receptor to which the parent ligand binds such that a covalent bond is formable between the conjugation agent and the receptor moiety. Also disclosed are compositions, probes and methods of detecting and/or quantifying receptors using the modified ligands of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/178,756, filed Jan. 28, 2000, the disclosure of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates generally to ligand-receptor interactions. Inparticular, the present invention relates to modified ligands that bindirreversibly to their cognate receptors, and to methods for preparingsuch ligands. The novel ligands of the invention have utility inter aliafor investigating protein function and as drugs (in healthcare,agricultural and environmental applications) for more effectivelyinhibiting or stimulating cognate receptor function.

BACKGROUND ART

Pharmacological receptors are intracellular or membrane-bound proteinswhich produce a pharmacological effect after binding with a specificligand. In this regard, a pharmacological receptor has a dual functionto (a) detect a ligand signal by forming a ligand-receptor complex andto (b) conduct and translate the signal leading to the pharmacologicaleffect.

Drugs can replace endogenous physiological ligands to interact withreceptors. A prerequisite for such a drug-receptor interaction is theformation of a drug-receptor complex, just as in the case ofligand-receptor interaction In contrast to physiological ligands thatstimulate an effect after binding to a receptor (receptor-mediatedeffects), drugs can be classified as (a) agonists or drugs whichstimulate an effect after binding to the receptor, and (b) antagonistsor drugs which do not stimulate an effect after receptor binding.

Several types of molecular interactions are possible for drug-receptorbinding including ionic bonds, hydrogen bonds, and hydrophobic bonds byvan der Waals forces. The vast majority of receptor interactions involveseveral kinds of binding simultaneously. Ionic bonds are important forthe primary phase of drug-receptor interaction since these bonds havethe greatest or longest range. After the initial interaction,fine-tuning takes place involving dipole-dipole-bonds, hydrogen bondsand hydrophobic bonds. Although all these interactions also fix the drugmolecule in the receptor's active site, the bindings are neverthelessreversible, as the force of interaction is very weak. Hence thepharmacological effectiveness of any drug is often affected by its ownconcentration in the plasma, as a decrease in plasma drug concentrationwill increase the dissociation of drug molecule from its receptor.

Several agents are known to inhibit enzymes irreversibly orpseudoirreversibly and the precise mechanism of inhibition gives rise tosubtle differences in the inhibition profiles and the duration ofinhibition.

Many inhibitors of acetylcholinesterase react covalently with thisenzyme to form an acyl enzyme that deacylates more slowly than theacetyl enzyme formed with the natural substrate acetylcholine. Theacetyl enzyme forms rapidly by attack of the active site serine on thesubstrate. Transfer of the acyl group to the enzyme occurs through atetrahedral intermediate. The acetyl enzyme is rapidly hydrolyzed, witha half time of 10 μsec. These rapid acylation and deacylation steps giverise to a turnover rate of 10⁵ substrate molecules per enzyme moleculeper second. Cholinesterase inhibitors such as physostigmine andneostigmine form methylaminocarbamyol and dimethylaminocarbamoylenzymes, which have half times for deacylation of several minutes. Thus,by providing the enzyme with an alternative substrate, catalysis ofacetylcholine is precluded during the catalytic cycle for thecarbamoylating agent. The kinetic constants for the respective acylationsteps for the acetoxy and carbamoxy ester substrates do not greatlydiffer, hence the longer residence time of the carbamoyl enzymeconjugate is an important factor in favoring inhibition.

Several other enzymes are inhibited by covalent attachment of theinhibitor, giving rise to irreversibility. The hydrazines (phenelzine,isocarxazid metabolites) and the acetylenic agents (pargyline) areoxidized to reactive intermediates by monoamine oxidase. Theseintermediates attack the associated flavin cofactor on the enzyme. Suchagents have been termed suicide substrates since their activationrequires catalysis by the very enzyme that they inactivate. Hence theinactivation process is mechanism-based. There are now many examples ofsuch substrates, activation of which by the enzyme results in covalentmodification of the enzyme or of an associated cofactor. Often thisoccurs by conjugation or association of the enzyme with its substratefollowed by a neighboring group attack. Several of the targets ofsuicide substrates have therapeutic significance. These include thepenicillinases and alanine racemases in antibacterial design; GABAtransaminase inhibitors for antiepileptic agents; lipoxygenase andcyclooxygenase inhibitors to control leukotriene and prostaglandinbiosynthesis, respectively, aromatase inhibitors to block formation ofestrogenic hormones; ornithine decarboxylase inhibitors as antiparasiticagents; and dopamine β-hydroxylase inhibitors to control catecholaminebiosynthesis. Many suicide substrates serve as antimetabolites and arepotential antineoplastic agents. The effectiveness of these inhibitorsdepends not only on their relative dissociation constants or K_(m)values compared with those of the endogenous substrate but also onkinetic competition between turnover of the suicide substrate and theinactivation event.

Omeprazole (PRILOSEC) is another well-known irreversible binding drugthat has been released for clinical use. This drug inhibits gastric acidsecretion by binding to the H⁺, K⁺-ATPase present only in the apicalmembrane of parietal cells. Omeprazole is especially useful in patientswith hypergastrinemia and may be valuable in those whose peptic ulcerdisease is not well controlled by H₂ antagonists. Omeprazole contains asulfinyl group in a bridge between substituted benzimidazole andpyridine rings. At neutral pH, this drug is a chemically stable,lipid-soluble, weak base that is devoid of inhibitory activity. Thisneutral weak base reaches parietal cells from the blood and diffusesinto the secretory canaliculi, where the drug becomes protonated andthereby trapped. Protonated drug rearranges to form a sulfenic acid anda sulfenamide. The sulfenamide interacts covalently with sulfhydrylgroup at critical sites in the extracellular (luminal) domain of themembrane-spanning H⁺, K⁺-ATPase. Omeprazole must thus be considered asprodrug that needs to be activated to be effective.

Despite the availability of several agents that bind their targetsirreversibly, there is a dearth of methods currently available forrationally designing ligands to irreversibly bind a target receptor.

DISCLOSURE OF THE INVENTION

The present invention arises, at least in part, from the unexpecteddiscovery that by attaching a conjugation agent to a parent ligand thatreversibly binds a target receptor, wherein the conjugation agent isreactive with a moiety of the target receptor such that a covalent bondis formable between the conjugation agent and the moiety, the modifiedligand thus produced is capable of binding the target receptorirreversibly.

Accordingly, in one aspect of the invention, there is provided a processfor modifying a parent ligand, comprising attaching to said parentligand a conjugation agent that is reactive with a moiety of a targetreceptor to which said parent ligand binds such that a covalent bond isformable between said conjugation agent and said moiety.

Suitably, the conjugation agent is attached to the parent ligand througha spacer.

Preferable, the spacer is covalently attached to the parent ligand.

Preferably, the spacer is covalently attached to the conjugation agent.

The spacer is suitably radical selected from the group consisting ofalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl,heterooxoalkyl, alkenyl, hetero alkenyl, aralkyl, hetero aralkyl, aryland heteroaryl radicals or any other molecular conformation which servesthe function of being a spacer. The length of the spacer arm is suitablyselected from a range of between about 0 Å and about 20 Å.

Preferably, the spacer is a non-hydrolysable radical under physiologicalconditions.

Preferably, the conjugation agent is selected from the group consistingof a sulfhydryl group specific conjugation agent, an amino groupspecific conjugation agent, a carboxyl group specific conjugation agent,a tyrosine specific conjugation agent, an arginine specific conjugationagent, a histidine specific conjugation agent, a methionine specificconjugation agent, a tryptophan specific conjugation agent, and a serinespecific conjugation agent.

The sulfhydryl group specific conjugation agent may be selected from thegroup consisting of N-maleimide, N-maleimide derivatives and disulfidereagents including, but not restricted to, 5′-dithiobis-(2-nitrobenzoicacid), 4,4′-dithiodipyridine, methyl-3-nitro-2-pyridyl disulfide, andmethyl-2-pyridyl disulfide.

The amino group specific conjugation agent may be selected from thegroup consisting of alkylating agents including, but not restricted to,α-haloacetyl compounds, aryl halides, aldehydes and ketones, andacylating agents including, but not restricted to, isocyanate,isothiocyanate, imidoesters, N-hydroxylsuccinimidyl ester, ρ-nitrophenylester, acyl chloride, and sulfonyl chloride.

The carboxyl group specific conjugation agent may be selected from thegroup consisting of carbodiimides and carboxyl group esterificationreagents including, but not restricted to, diazoacetate esters anddiazoacetamides.

The tyrosine specific conjugation agent may be selected from diazoniumderivatives including, but not limited to, benzidine and bis-diazotized3,3′-dimethylbenzidine.

The arginine specific conjugation agent may be selected from1,2-dicaxbonyl reagents including, but not restricted to, glyoxal,phenylglyoxal, 2-3-butanedione and 1,2-cyclohexanedione.

The histidine specific conjugation agent is suitably selected from thegroup consisting of alkylating agents including, but not restricted to,α-haloacetyl compounds, aryl halides, aldehydes and ketones, andacylating agents including, but not restricted to, diethylpyrocarbonate,ethoxyformic anhydride, isocyanate, isothiocyanate, imidoesters,N-hydroyxlsuccinimidyl ester, ρ-nitrophenyl ester, acyl chloride, andsulfonyl chloride.

The methionine specific conjugation agent may be selected from the groupconsisting of alkylating agents including, but not restricted to,α-haloacetyl compounds, aryl halides, aldehydes and ketones.

The tryptophan specific conjugation agent may be selected from the groupconsisting of N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide andρ-nitrophenylsulfenyl chloride.

The serine specific conjugation agent may be selected from the groupconsisting of diisopropylfluorophosphate and acrylsulfonyl fluoridesincluding, but not restricted to, phenylmethyl-sulfonylfluoride.

The parent ligand may be any natural or non-natural ligand but ispreferably a biologically active ligand inclusive of known drugs andnaturally occurring or synthesized drug candidate compounds.

In another aspect, the invention provides a modified ligand produced bythe process broadly described above.

In yet another aspect of the invention, there is provided a modifiedligand having the general formula:L-R₁-A   (I)

wherein L is a parent ligand;

wherein A is a conjugation agent that is reactive with a moiety of atarget receptor to which the parent ligand binds such that a covalentbond is formable between said conjugation agent and said moiety, and

R₁ is an optional spacer which preferably comprises a non-hydrolysableradical selected from the group consisting of alkyl heteroalkyl,cycloalkyl, heterocycloalkyl oxoalkyl, heterooxoalkyl, alkenyl, heteroalkenyl, aralkyl hetero aralkyl, aryl and heteroaryl radicals.

In a preferred embodiment, the modified ligand is interactive with esnucleoside transporter and/or nucleoside/nucleotide/nucleobase-sensitiveproteins, wherein said modified ligand has a general formula selectedfrom the group consisting of:

wherein A is N-maleimide, 2-pyridyldithio, or halogen; X is NH, S, or O;Y is H, halogen, NH₂, or O; Z is H, halogen, or CH₃; R₁ is spacer armcomprising a non-hydrolysable radical preferably under physiologicalconditions; R₂ is H, β-D-ribose, β-D-2-deoxyribose, or their 5′-mono-,5′di-, and 5′tri-phosphate.

Suitably, the modified ligand inhibits said es nucleoside transporterand/or nucleoside/nucleotide/nucleobase-sensitive proteins and has ageneral formula selected from the group consisting of:

wherein R₄ is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], orN-[ε-caproylate];

wherein R₄ is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], orN-[ε-caproylate];

wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or6-[3′-propioamido]hexanoate; and

wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or6-[3′-propioamido]hexanoate.

In another embodiment modified ligands that bind to seratonin receptorscan be used.

In another aspect, the invention resides in a composition comprising themodified ligand as broadly described above, together with apharmaceutically acceptable carrier.

In a further aspect of the invention, there is provided a method oftreatment or prophylaxis of a condition associated with a targetreceptor, said method comprising administering to a patient in need ofsuch treatment a therapeutically effective dosage of the composition asbroadly described above.

According to another aspect of the invention, there is provided a methodof detecting the presence of a target receptor in a test sample,comprising: contacting said sample with a modified ligand as broadlydescribed above, wherein said modified ligand binds said targetreceptor, and detecting the presence of a complex comprising saidmodified ligand and said receptor in said contacted sample.

In another aspect of the invention, there is provided a method ofquantifying the presence of a target receptor in a test sample,comprising: contacting said sample with a modified ligand as broadlydescribed above, wherein said modified ligand binds said targetreceptor; measuring the concentration of a complex comprising saidmodified ligand and said receptor in said contacted sample; and relatingsaid measured complex concentration to the concentration of saidreceptor in said sample.

In yet another aspect, the invention provides a method of detecting thepresence of a target receptor on a cell or cell membrane, comprising:contacting a sample containing said cell or cell membrane with amodified ligand as broadly described above, wherein said modified ligandbinds said target receptor; and detecting the presence of a complexcomprising said modified ligand and said cell or cell membrane in saidcontacted sample.

In another aspect of the invention, there is provided a method ofquantifying the presence of a target receptor on a cell or cellmembrane, comprising: contacting a sample containing said cell or cellmembrane with a modified ligand as broadly described above, wherein saidmodified ligand binds said target receptor, measuring the concentrationof a complex comprising said modified ligand and said cell or cellmembrane in said contacted sample; and relating said measured complexconcentration to the concentration of said receptor present on said cellor cell membrane.

In another aspect, the invention extends to a probe that covalentlybinds to a target receptor, said probe comprising a modified ligand asbroadly described above having a reporter molecule associated therewith.

In one embodiment, the probe comprises the modified ligand that isinteractive with es nucleoside transporter and/ornucleoside/nucleotide/nucleobase-sensitive proteins, as broadlydescribed above.

In this respect, the cell is preferably an animal cell, more preferablya mammalian cell and more preferably a human cell. Alternatively, thecell may be a plant cell or a microbial cell. The microbial cellincludes, but is not restricted to, a cell of bacterial, viral or fungalorigin.

The invention also encompasses the use of the modified ligand and probeas broadly described above inter alia in the study, treatment andprevention of conditions associated with their corresponding targetreceptors.

In one embodiment, there is provided process for modifying a parentligand, comprising attaching to said parent ligand a conjugation agentthat is reactive with a moiety of a target receptor to which said parentligand binds, wherein when said parent ligand binds to the receptor acovalent bond is formed between said conjugation agent and said moiety.

In one preferred embodiment, the conjugation agent is positioned on theligand at a position that promotes and/or permits covalent bondformation with the moiety of the target receptor. In another preferredembodiment, the receptor to which the ligand binds is an active siteassociated with a biological activity. The receptor is, for example, acell surface receptor. The binding of the modified ligand to thereceptor in one embodiment is associated with altered activity of thetarget receptor.

In another preferred embodiment, the parent ligand and/or receptor arenaturally occurring. In another embodiment, the modified ligand is not acrosslinking agent. In another embodiment, the modified ligandoptionally does not comprise a photo-reactive group such as aphotolabel. In another embodiment, the modified ligand does not comprisea label. In another embodiment, the receptor to which the ligand bindsis not a nucleic acid, such as RNA or DNA. In another embodiment, theconjugation agent does not bind to a moiety of a nucleic acid, andoptionally binds to a residue of an amino acid.

In one embodiment, there is provided process for modifying a parentligand, comprising attaching to said parent ligand a conjugation agentthat is reactive with a moiety of a target receptor to which said parentligand binds, wherein when said parent ligand binds to the receptor acovalent bond is formed between said conjugation agent and said moiety,and wherein the parent ligand binds specifically with a nucleosidetransporter.

In another embodiment there is provided a process for modifying a parentligand, comprising attaching to said parent ligand a sulfhydryl groupspecific conjugation agent that is reactive with a sulfhydryl group of atarget receptor to which said parent ligand binds, wherein when saidparent ligand binds to the receptor a covalent bond is formed betweensaid conjugation agent and said sulfhydryl group, and wherein the parentligand binds specifically with a serotonin receptor.

In one embodiment, there is provided a modified ligand having thegeneral formula:L-R₁-A   (I)

wherein L is a parent ligand that binds specifically with a targetreceptor comprising a nucleoside transporter,

wherein A is a conjugation agent that is reactive with a moiety of thetarget receptor to which the parent ligand binds, such that when saidparent ligand binds to the receptor a covalent bond is formed betweensaid conjugation agent and said moiety, and R₁ is an optional spacer.

In another embodiment, there is provided a modified ligand having thegeneral formula:L-R₁-A   (I)

wherein L is a parent ligand that binds specifically with a targetserotonin receptor,

wherein A is a conjugation agent that is reactive with a sulfhydrylgroup of said target receptor to which the parent ligand binds, suchthat when said parent ligand binds to the receptor a covalent bond isformed between said conjugation agent and said sulfhydryl group of saidreceptor, and R₁ is an optional spacer.

The disclosure of all patents, patent applications, publications andpublished patent applications referred to herein are incorporated hereinby reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention, as exemplified by preferred embodiments, is describedwith reference to the following drawings in which:

FIG. 1 shows the NEM inhibition of equilibrative ³H-uridine transport inmurine myeloma SP2/0-Ag14 cells.

FIG. 2 shows the effects of NEM on the kinetics of ³H-NBMPR equilibriumbinding in murine myeloma SP2/0-Ag14 cells.

FIG. 3 shows the general reaction scheme for synthesis of CrMCC.

FIG. 4 shows the rate of CrMCC formation.

FIG. 5 shows the light absorbance profile of CrMCC and NBMPR.

FIG. 6 shows the inhibition of ³H-NBMPR binding by CrMCC, cytidine andSMCC in human HL-60 promyelocytic leukemia plasma membranes.

FIG. 7 shows the effect of CrMCC on the kinetics of ³H-NBMPR binding tohuman HL-60 promyelocytic leukemia plasma membranes.

FIG. 8 shows the effects of CrMCC, cytidine and SMCC on growth of HL-60cells.

FIG. 9 shows the time course of ³H-CrMCC binding to human HL-60promyelocytic leukemia plasma membranes.

FIG. 10 shows the dissociation of ³H-CrMCC and 3H-cytidine from thebinding sites of human HL-60 promyelocytic leukemia plasma membranes.

FIG. 11 shows the concentration dependence of 3H-CrMCC binding to humanHL-60 promyelocytic leukemia plasma membranes.

FIG. 12 shows the effect of pH on the dissociation of ³H-CrMCC from itsbinding site in human HL-60 promyelocytic leukemia plasma membranes.

FIG. 13 shows the inhibition of ³H-CrMCC binding to human HL-60promyelocytic leukemia plasma membranes.

FIG. 14 shows the covalent binding of sulfhydryl reactive ³H-cytidineanalogs to human HL-60 promyelocytic leukemia plasma membranes.

FIG. 15 shows the UV absorbance profile of reversed-phase chromatographyof human HL-60 promyelocytic leukemia plasma membrane proteins.

FIG. 16 shows the radioactivity profile of reversed-phase chromatographyof human HL-60 promyelocytic leukemia plasma membrane proteins.

FIG. 17 shows the covalent binding of sulfhydryl group reactive³H-adenosine analogs to human HL-60 promyelocytic leukemia plasmamembranes.

FIG. 18 shows the inhibition of ³H-5-HT binding to murine brainmembranes by various 5-HT analogs.

FIG. 19 a shows the chemical structure of LBT3001(1-[2-5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione)).

FIG. 19 b shows-the reaction scheme for synthesis of LBT3001.

FIG. 20 a shows the chemical structure of LBT3002(4-2,5-dioxo-2,5-dihydro-pyrrol-1-yl)N-[2-(5-hydroxyl-1H-indol-3-yl)-ethyl]-butyramide).

FIG. 20 b shows the reaction scheme for synthesis of LBT3002.

FIG. 21 a shows chemical structure of LBT3004(3-2,5dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-5-hydroxyl-1H-indol-3-yl)-ethyl]-propionamide).

FIG. 21 b shows the reaction scheme for synthesis of LBT3004.

FIG. 22 a shows the chemical structure of LBT3005(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexane carboxylicacid [2-5-hydroxy-1H-indol-3-yl)-ethyl]-amide).

FIG. 22 b shows the reaction scheme for synthesis of LBT3005.

FIG. 23 shows the structures of exemplary ligands that can be modifiedto include a conjugation agent.

DETAILED DESCRIPTION

1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “attached” is meant direct or indirect attachment of a conjugationagent to a ligand in such a manner as to resist separation of theconjugation agent from the ligand under normal physiological conditions.Accordingly, the term “attached” as used herein includes within itsscope one more ionic bonds, hydrogen bonds, van der Waals forces,covalent bonds or combinations thereof that form between the conjugationagent and the ligand or between an intervening spacer and theconjugation agent and the ligand, respectively, such that separation ofthe conjugation agent from the ligand is resisted under normalphysiological conditions.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “conjugation agent” is meant a moiety of a modified ligand that isreactive with a moiety of a receptor that binds a parent ligand fromwhich the modified ligand was derived wherein a covalent bond isformable between the conjugation agent and the receptor moiety. In thisconnection, the conjugation agent may be sufficient either on its own orin the presence of an ancillary conjugation agent to facilitate covalentcoupling with the receptor moiety. The ancillary coupling agent may bean enzyme that catalyzes, or an activating agent that causes, formationof a covalent bond between the conjugation agent and said receptormoiety.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state.

As used herein, the term “ligand” refers to an agent that binds,interacts or otherwise associates with, a target receptor. The agent maybind the target receptor when the target receptor is in its nativeconformation, or when it is partially or totally unfolded or denatured.According to the present invention, a ligand is not limited to an agentthat binds a recognized functional region of the target receptor e.g.the hormone-binding site of a receptor, and the like. In practicing thepresent invention, a ligand can also be an agent that binds any surfaceor internal sequences or conformational domains of the target receptor.Preferably, the ligand is a molecule affecting physiological functionincluding a drug. The ligand can also be an endogenous ligand.

By “obtained from” is meant that a sample such as, for example, anextract comprising a receptor is isolated from, or derived from, aparticular source such as a suitable cell or tissue source inclusive ofhuman, animal plant or microbial origin.

The term “patient” as used herein refers to any organism in whichtherapy or prophylaxis of a condition associated with a receptor isdesired using the methods of the invention. However, it will beunderstood that “patient” does not imply that symptoms are present. Thepatient may therefore include a microbe, plant or animal. Preferably,the patient is a human or other mammal and includes any individual it isdesired to examine or treat using the methods of the invention. Suitablemammals that fall within the scope of the invention include, but are notrestricted to, primates, livestock animals (e.g., sheep, cows, horses,donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats,guinea pigs, hamsters), companion animals (e.g. cats, dogs) and captivewild animals (e.g., foxes, deer, dingoes).

By “pharmaceutically acceptable carrier” is meant a solid or liquidfiller, diluent or encapsulating substance that may be safely used intopical or systemic administration.

The term “pharmaceutically acceptable salts” as used herein refers tonon-toxic salts of the modified ligands of this invention, which aregenerally prepared by reacting the free base with a suitable organic orinorganic acid. Representative salts include the following salts:acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,borate, bromide, calcium edetate, camsylate, carbonate, chloride,clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate,esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,oleate, oxalate, pamaote, palmitate, panthothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide, valerate.

The term “receptor” as used herein refers to a structure including amolecule or a cluster of molecules that is specific for one or moreligands wherein binding, interaction or otherwise association of theligand(s) with the receptor effects, changes or nullifies a finction ofthe receptor. The receptor is preferably, but not exclusively, aprotein. Representative receptors include, but are not limited to, aninsulin receptor, epidermal growth factor receptors, γ-aminobutyric acidreceptors, nicotinic acetylcholine receptors, serotonin receptors, α-and β-adrenoceptors, dopamine receptors, histanine receptors, prostanoidreceptors, adenosine receptors, cyclic nucleotide recetors, glutamatereceptors, cytoktne receptors, atrial natuetic peptide receptors, andthe prostaglandin receptors. The receptor can include transporters suchas glucose, amino acids transporters, sodium-proton exchangers,chloride-bicarbonate exchangers, sodium pumps, calcium pumps, protonpumps; channels such as sodium channels, potassium channels, calciumchannels and chloride channels; enzymes such as oxidoreductases,transferases, hydrolases, lyases, isomerases, and ligases. However, itwill be understood that the receptor need not be a protein and mayinclude, for example, a nucleic acid in which the amino group found onadenine, guanine and cytosine may be targeted by a conjugation agentaccording to the invention. Alternatively, the receptor may be acarbohydrate (e.g., amino-containing carbohydrates such as aminophenylglycosides) or a lipid (e.g.. present on the phosphate head groups ofsome phospholipids) having one or more carboxyl groups, and/or one ormore amino groups that may be targeted by a conjugation agent.

By “reporter molecule” as used in the present specification is meant amolecule that, by its chemical nature, provides an analyticallyidentifiable signal that allows the detection of a complex comprising aligand and its cognate receptor. The term “reporter molecule” alsoextends to use of cell agglutination or inhibition of agglutination suchas red blood cells on latex beads, and the like.

The term “sample” as used herein refers to any suitable sample that maycontain a target receptor according to the invention. The sample may beextracted, untreated, treated, diluted or concentrated from any suitablesource and may contain one or more cells and/or cell membranes. Thesample may comprise whole cells, denatured cells, cellular membranes orparts thereof. Alternatively, the sample may contain an isolatedreceptor. Suitably, the sample may comprise cells obtained from a tissuebiopsy. Alternatively, the sample may comprise cells or cell lines,which have been cultured in vitro.

The term “spacer” as used herein refers to a chemical linker, polymer,peptide and the like that spatially separates the conjugation agent fromthe ligand. Preferably, the spacer is selected such that it does notinterfere with the binding of the modified ligand to the receptor.

By “therapeutically effective amount”, in the context of the treatmentof a condition associated with a receptor, is meant the administrationof that amount to a patient in need of such treatment, either in asingle dose or as part of a series, that is effective for treatment ofthat condition. The effective amount will vary depending upon the healthand physical condition of the individual to be treated, the taxonomicgroup of individual to be treated, the formulation of the composition,the assessment of the medical situation, and other relevant factors. Itis expected that the amount will fall in a relatively broad range thatcan be determined through routine trials.

2. Modified Ligands

The present invention resides, at least in part, in the surprisingdiscovery that a conjugation agent can be attached to a parent ligand toform a modified ligand that binds irreversibly to a target receptor towhich the parent ligand binds reversibly. The irreversible binding ofthe modified ligand to the receptor is effected by formation of acovalent bond between the conjugation agent and a moiety present on thereceptor, which is preferably one or more functional amino acid sidechain groups (sometimes referred to herein as “functional groups”). Thecovalent bond is formed by association of the modified ligand with thereceptor, followed by neighboring reactive functional group attack bythe conjugation agent. This irreversible interaction of the modifiedligand with the receptor results in either permanent inhlibition (for anantagonist) or stimulation (for an agonist) of the receptor functions.Normal function or activity of the receptor resumes only after newreceptors are synthesized.

Parent ligands that can be modified include ligands that are interactivewith es nucleoside transporter and/ornucleoside/nucleotide/nucleobase-sensitive proteins. Two forms ofnucleoside transporters are classified based on their sensitivity toinhibition by NBMPR (nitrobenzylthioinosine). See Griffith et al.,Biochim. Biophys. Acta, vol. 1286, pp. 153-181 (1996). The group oftransporters sensitive to NBMPR is designated es (equilibriumsensitive), and the group insensitive is designated ei (equilibriuminsensitive). In one embodiment, compounds are provided that havedifferential binding to es vs. ei receptors. Compounds are provided thatbind selectively and irreversibly to es receptors. The expression of esreceptors is positively correlated with the carcinogenic state of cells.

Among the numerous reactive functional amino acid side chains on the esnucleoside trasporter proteins that can be used for covalent attachment,the sulfflydryl group of cysteine residue is probably the mostpharmacological and biologically important. Early studies on the effectof sulfhydryl reagents on nucleoside transport in mammalian cells hadshown to cause a marked inhibition of uridine uptake in a variety ofcultured cells (Plagemann & Richey (1974), Biochim. Biophs. Acta, vol.344, pp. 263-305; Plagemann et al. (1978), J. Cell. Physiol., vol. 97,pp. 49-72; Plagemann & Wohlhueter (1980), Curr. Top. Membr. Transp.,vol. 14, pp. 225-230; Belt (1983), Biochem. Biophys. Res. Commun., vol.110, pp. 417-423; Belt (1983), Mol. Pharmacol., vol. 24, pp. 479-484).However most of the earlier studies were directed toward the totalnucleoside uptake and little attempt were made to distinguish thedifferential in sensitivity towards sulfhydryl reagents by the es and eitrinsport systems. Furthermore, the sulfhydryl reagents used were mainlyof organomercurial compounds, which were demonstrated to perturb plasmastructure at concentration as low as 200 μM (Belt & Noel (1985). BiochemJ., vol. 232, pp.681-688).

Ligands also may be modified which are ligands that bind to serotonin(5-hydroxytryptamine, or 5-HT) receptors. 5-HT receptors include adiversity of receptor subtypes (Peroutka, S. J., CNS Drugs, (1995), vol.4 (Suppl 1), pp. 18-28). With the exception of the 5-HT₃ receptor, whichis an ion channel (Derkash, V. et.al, Nature, (1989), vol. 339, pp.706-709), other 5-HT receptors belong to the extensive family of seventransmembrane G protein-coupled receptors. The clinical significance ofthe effects of 5-HT is manifest for example, in neurological, CNS,psychiatric and mood disorders, includimg migraine, anxiety, depression,schizophrenia, obsessive compulsive disorder, psychosis, aggression,hostility, eating disorders, gastrointestinal disorders, hypertension,the maintenance of the circadian rhythms in the sleep-wakefulness cycle,sexual activity, compulsive behavior, temperature, emesis, andcardiovascular and motor function. Drugs that target the 5-HT receptorsthus have wide clinical applications. Ligands that bind to 5-HTreceptors can have effects by binding, for example, to thecardiovascular system, platelets, gastrointestinal tract, and the brain(Erspamer, V, Ed. “5-Hydroxytryptamine and Related Indolealklylamines”,Handbuch der Expermentellen Pharnakologie, Vol. 19. Springer-Verlag,Berlin, (1996), pp. 132-181).

Thus, for example, the ligand 5-HT may be modified as disclosed herein.Other ligands that can be modified include 5-HT precursors and 5-HTreceptor agonists and antagonists known in the art. Examples include theprecursor 5-hydroxytryptophan, which has been used as an antidepressantdrug; 5-HT_(1A)-agonists used as tranquilizers and antihypertensives;sumatriptan, a selective 5-HT₁ receptor agonist, used for migraine;5-HT₂-antagonists such as methysergide, used for migraine prophylaxisand in carcinoid tumour syndrome; 5-HT₂-antagonists such as ketaserin,used to lower the blood pressure in hypertensive patients; and selective5-HT₃-antagonists used to treat cytostatic- and radiation-inducedemesis. (Beasley, C. M., et.al, Psychopharmacology, (1992), vol. 107,pp. 1-10; Bolden-Watson, C, and Richelson, E., Life Sciences, (1993),vol. 52, pp. 1023-1029.; Koe, B. K., J. Clin. Psychiatry, (1990), vol.51, pp. 13-17).

Serotonin binding ligands which can be modifed include those developedfor CNS disorders, such as anxiety, such as benzodiazepines, or⁵-HT_(1A) agonists, such as buspirone, gepirone, ipsapirone, andflesinoxan. As anxiolytic agents, they possess advantages overbenzodiazepines because they lack the sedation and drug dependenceliabilities of bezodiazepines (Barrett, J. E., and Vanover, K. E.,Psychopharmacology, (1993), vol. 112, pp. 1-12.). Other serotoninbinding ligands include those developed for treatment of depression,such as SSRIs, for example fluoxetine. SSRIs have also been shown tohave efficacy in the treatment of bulimia and obsessive-compulsivedisorders and may be also useful in treating obesity, panic disorders,premenstrual syndrome, diabetic neuropathy, chronic pain, and certaincognitive aspects of Alzheimer's disease. Other useful antidepressantdrugs which are relatively potent antagonists at 5-HT₂ receptors,include tricyclic antidepressants, such as trazadone and nefazodone(Cowen, P. J, and Anderson. I. M., “5-Hydroxytryptamine in Psychiatry”(Sandler, M., Coppen, A., & Harnett, S. Eds.), .Oford University Press,New York; (1991)). Ligands include compounds developed for the treatmentof schizoprenia, including the antipsychotic agents clozapine,risperidone, and olanzepine, (Weil-Malherbe, H., Serotonin andschizophrenia. In “The Central Nervous System. Vol. 3, Serotonin inHealth and Disease”, Essman, W. B., Ed, Spectrum Publications, Inc., NewYork; (1978), pp. 231-291). Ligands associated with the treatment ofeating disorders can be used, such as the SSRIs and fenfluramine.Fenfluramine may be useful in the treatment of other diseases, such asautism, premenstrual syndrome, seasonal affective disorder, andattention deficit disorder. Ligands associated with alleviation ofgastrointestinal disorders can be used, such as ondansetron andgraniseton, which are used in the treatment of radiation induced emesisassociated with cancer chemotherapy, and for ameliorating the nausea andvomiting occurring during recovery from general anesthesia. Otherligands include metoclopramide and cisapride. Ligands used in control ofthe sleep-wakefulness cycle, such as L-tryptophan or nonselective 5-HTagonists or 5-HT antagonists such as ritanserin may be used (for areview, see Wauquier, A., and Dugovic, C., Ann N.Y. Acad. Sci., (1990),vol. 600, pp. 447-459). Other ligands include etanserin, a 5-HT₂receptor antagonist used as an antihypertensive agent.

2.1. Considerations Relating to Protein Structure Reactivity

Peptides and proteins are composed of amino acids polymerized togetherthrough the formation of peptide (amide) bonds. The peptide-bondedpolymer forms the backbone of polypeptide structure. There are 20 commonamino acids found throughout nature, each containing an identifying sidechain of particular chemical structure, charge, hydrogen bondingcapability, hydrophilicity (or hydrophobicity), and reactivity. The sidechains do not participate in peptide formation and are thus free tointeract and react with their environment.

Amino acids may be grouped by type depending on the characteristics oftheir side chains. The most significant amino acids for covalentconjugation purposes are the ones containing accessible ionizable sidechains such as aspartic acid, glutamic acid, lysine, arginine,histidine, cysteine, and tyrosine. Methionine and tryptophan alsocontaining ionizable side chains, however, they are not easilyaccessible as they are usually buried deep inside the molecularstructure of receptors due to their hydrophobic nature.

Both aspartic and glutamic acids contain carboxylate groups that haveionization properties. Carboxylate groups in proteins may be derivatizedthrough the use of amide bond forming agents or through active ester orreactive carbonyl intermediates.

Lysine, arginine, and histidine have ionizable amine containing sidechains. These amine containing side chains typically are exposed on thesurface of proteins and can be derivatized with ease. The most importantreactions that can occur with these residues are alkylation andacylation.

Cysteine is the only amino acid containing a sulfhydryl group. The mostimportant reaction of cysteine groups in proteins is the formation ofdisulfide cross-links with another cysteine molecule. Cysteinesulfhydryls and cystine disulfides (called cystine residues) may undergoa variety of reactions, including alkylation to form stable thioetherderivatives, acylation to form relatively unstable thioesters, and anumber of oxidation and reduction processes. Cysteine and cystine groupsare relatively hydrophobic and usually found within the core of aprotein. It is often difficult to access the sulfhydryl groups of largeproteins without the presence of a deforming agent or a “driver”.Nevertheless, such steric hindrance does give the sulfhydryl groups aleading edge in selectivity. Thus, to access the functional reactivesulfhydryl group situated deep into the ligand-binding site, withoutusing deforming agents, is to utilize its physiological ligands or drugsto direct the conjugation agent as herein described into the innerstructure of receptors. It is possible that irreversible binding drugsthat are targeted at the sulfhydryl groups is likely to have a lowerlevel of non-specific binding, in clinical term: “fewer side effects”,than any other functional groups.

Tyrosine contains a phenolic side chain. Although the amino acid is onlysparingly soluble in water, the ionizable nature of the phenolic groupsometime makes it appear in hydrophilic regions of a protein—usually ator near the surface. Thus unlike cysteine residue, tyrosinederivatization proceeds without much need for deforming agents tofurther open the protein structure. Tyrosine may be targetedspecifically for modification through its phenolate anion by acylation.

In summary, protein molecules may contain up to nine amino acids thatare readily derivatizable at their side chains. These nine residuescontain eight principal functional groups with sufficient reactivity formodification reactions. They are the guanidinyl group of arginine, theγ- and β-carboxyl groups of glutamic and aspartic acids, respectively,the sulfhydryl group of cysteine, the imidazolyl group of histidine, theε-amino group of lysine, the thioether moiety of methionine, the indolylgroup of tryptophan and the phenolic hydroxyl group of tyrosine. Sincemethionine and tryptophan are generally buried in the interior ofproteins and are thereby protected from conjugates dissolved in thesolvent, they show only some selected reactivity in intact proteins. Theother ionizable groups are normally either exposed on the surface ofproteins or can be accessed with the help of deforming agents or“drivers”. They are therefore the easy targets for conjugation. However,among the numerous reactive functional amino acid side chains on theproteins that can be used for labeling, the sulfhydryl group of cysteineresidue is most probably both pharmacologically and biologicallyimportant. It has been reported that in most macromolecules, there is atleast one copy of reactive sulfhydryl group situated at or closed to theligand binding sites of target macromolecules. Disruption of thisreactive sulfhydryl functional group by sulfhydryl reducing agents hasbeen shown to affect the functionalities of many macromolecules.

2.2. Moieties of Receptors Permitting Conjugation

The sulfhydryl moiety, with the thiolate ion as the active species, isthe most reactive functional group in a protein. With a pK_(a) of about8.6, the reactivity of the thiol is expected to increase with increasingpH, toward and above its pK_(a).

In the process of modifying a parent ligand, it is advantageous tocapitalize the presence of this highly reactive sulfhydryl group, whichis typically situated near or at the ligand-binding site of thereceptor. Drugs or physiological ligands can be chemically modified toinclude a conjugation agent that reacts faster with the thiol group thanany other reactive functional groups. Upon association of this modifieddrug with its receptor, this sulfilydryl group directed conjugate wouldattack any sulfhydryl group that is situated within its reachableproximity, this causes covalent binding of the drug to its receptor.

In addition to sulfhydryl group, there are also other highly reactivefunctional groups present on the amino acid side chains, which can bechemically modified. Conjugates that are reactive to these functionalgroups will be discussed below.

2.3. Conjugation Agents

2.3.1. Sulfhydryl Group Specific Conjugation Agents

N-Maleimide derivatives. Maleimides are considered fairly specific tothe sulfhydryl group, especially at pHs below 7 where other nucleophilesare protonated. In acidic and near neutral solutions, the reaction ratewith simple thiols is about 100-fold faster than with the correspondingsimple amines. Although the rate increases with pH, the reaction withthe amino group also becomes significant at high pHs. The other majorcompeting reaction is the hydrolysis of maleimides to maleamic acids.However, at pH 7, the apparent rate of hydrolysis is only 3.2×10⁴ min⁻¹in 0.1 M sodium phosphate buffer at 20° C., which is too slow tointerfere with the reaction with sulfhydryl groups., The rate ofdecomposition becomes significant only at pH above neutrality. Thiolundergo Michael reaction with maleimides to yield exclusively the adductto the double bond. The resulting thioether bond is very stable andcannot be cleaved under physiological conditions.

Disulfide reagents. Disulfide interchange occurs when sulfhydryl groupsreact with disulfides. Some of the most commonly used disulfide reagentsare 5,5′-dithiobis-(2-nitrobenzoic acid), 4,4′-dithiodipyridine,methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl disulfide. Theprotein disulfides formed are readily reverse in the presence of freemercaptan such as 2-mercaptoethanol or dithiothreitol. The reduction ofprotein disulfide into its original sulfhydryl group allows the proteinto regain its functions. Thus, irreversible binding drugs of thiscategory provide additional safety mechanism to counter varioustherapeutic complications such as over-dosing, hyper reaction, etc.

2.3.2. Amino Group Specific Conjugation Agents

The amino group is another strong nucleophile in the protein. However,because of its abundance and omnipresence in proteins, and therelatively high pK_(a) of the ammonium ion, most of the reagents thatreact with the amino group can also react with other functionalities.Thus, it may not be an ideal target for modified ligands according tothe invention, unless the critical cysteine residues are absent from thedrug-binding site. Nevertheless, many stable acylated products are stilland only formed with the amino groups. The most common reactions ofamines are alkylation and acylation reactions. Some of the importantalkylating agents that can be incorporated into ligand structures are:α-haloacetyl compounds (e.g., haloacetate, haloacetamides), N-maleimidederivatives, aryl halides (e.g., dinitrofluorobenzene,trinitrobenzenesulfonate), aldehydes (e.g., glutaraldehyde,formaldehyde) and ketones (e.g., pyridoxal phosphate). Acylating agentsinclude, but are not restricted to, isocyanate, isothiocyanate,imidoesters, N-hydroyxlsuccinimidyl ester, ρ-nitrophenyl ester, acylchloride, and sulfonyl chloride.

2.3.3. Carboxyl Group Specific Conjugation Agents

The most important chemical modification reactions of carboxyl groupsutilize the carbodiimide-mediated process. With proteins, the optimum pHof the reaction is about 5, which is difficult to achieve in mostphysiological conditions. Other reagents such as diazoacetate esters anddiazoacetamides can also be used to esterify carboxyl groups. Similar tocarbodiimides, these reagents react with high specificity with carboxylgroups of proteins under mild acid conditions.

2.3.4. Tyrosine Specific Conjugation Agents

Tyrosine, histidine, and other aromatic residues of proteins are rich inelectrons. These residues undergo electrophilic substitution reactionsat the aromatic ring. Useful electrophiles for reaction with tyrosineand histidine in proteins are diazonium compounds. Other proteincomponents such as lysine, tryptophan, cysteine, and arginine residuesreact very slowly, such that diazonium reagents can be regarded astyrosine selective. Diazonium ions are generally unstable even atneutral pH and maximum reaction with the proteins is typically achievedat alkaline pH. The phenolate ion of tyrosine also reacts similar toamino groups toward acylating agents. However, the tyrosyl group isgenerally perceived as having a lower reactivity. This is not becausethe phenolate ion has lower nucleophilicity, but because tyrosineresidues are usually buried in a protein and are, therefore, generallyinaccessible for reactions due to their hydrophobicity.

2.3.5. Arginine Specific Conjugation Agents

A predominant reaction of the guanidinyl moiety of arginine residues iswith 1,2-dicarbonyl reagents. Commonly used vicinal diketones includeglyoxal, phenylglyoxal, 2-3-butanedione and 1,2-cyclohexanedione.

2.3.6. Histidine Specific Conjugation Agents

While a number of alkylating agents react with the imidazolyl moiety ofhistidines have been referred to earlier, the rate of these reactions isgenerally inferior to other nucleophiles. Even with α-haloacetate,N-carboxymethylation is generally slow in comparison with sulfhydrylgroups. However, when such reactive α-halocarbonyl group is incorporatedinto affinity labels (e.g., p-toluenesulfonylphenylaninechloromethylketone, p-toluenesulfonyllysinechloromethylketone), specificreaction may be achieved. Beside α-haloacetyl groups, other alkylatingagents are not as reactive towards histidine. With acylating reagents,histidine forms acylated products that are generally unstable and mayundergo spontaneous hydrolysis. The most important acylating agent thathas been commonly used for the modification of histidines isdiethylpyrocarbonate or ethoxyformic anhydride. The acylated imidazoleis reversed at alkaline pH, resulting in the recovery of histidine.Deacylation can be achieved at neutral pH very rapidly withhydroxylamine.

2.3.7. Methionine Specific Conjugation Agents

The major chemical modification reaction of methionine is alkylation.Because methionine is often situated in the hydrophobic interior ofproteins, it tends to provide high degree of selectivity. Onlyalkylating reagents that are coupled to the ligands are accessible tothese buried methionine residues.

2.3.8. Tryptophan Specific Conjugation Agents

Due to its hydrophobicity, tryptophan residues are generally buried inthe interior of proteins. Tryptophan residues can be modified withN-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. A distinctreagent, ρ-nitrophenylsulfenyl chloride, has been used for modificationof the indolyl moiety. The reaction is selective for tryptophan andcysteine residues.

2.3.9. Serine Specific Conjugation Agents

Many reactive reagents such as diisopropylfluorophosphate,phenylmethylsulfonylfluoride and other acrylsulfonyl fluorides have beenfound to react with the active-site serine. Care should be exercised inuse of such reagents because of the strong competitive reaction ofhydrolysis.

2.4. Design of Modified Ligands of the Invention

The covalent bond formation between the modified ligand and the receptorcan be catalyzed by enzymes, caused by activating agents, or facilitatedby the conjugation agent on its own. The covalent bond is preferablyformed with a functional group situated at or near the ligand-bindingsite of the receptor. This strategy is advantageous as it ensures a highdegree of specificity.

The design of an irreversible binding ligand depends on the chemical,biological and molecular properties of both ligand and receptor. In eachinstance, the conjugation agent to be introduced onto the chemicalstructure of the ligand may be different and may require a certainconfiguration. In general, the ligand preferably includes a functionalgroup which permits attachment of a conjugation agent, or which iscapable of modification to contain such a group, without affecting theactivity of the ligand to bind its receptor and to elicit a biologicalactivity. The modified ligand in this regard need not have the samebiological activity as the parent ligand (e.g., it may not require to beactivated in ivto by some metabolic or catabolic step).

Some of the conditions and requirements to be considered for selectionand configuration of the conjugation agent are as follows:

1. Determine reaction specificity towards a particular functional groupof the receptor that is required for selection of the conjugation agent,e.g., amino, sulfhydryl, carboxyl guanidinyl, imidazolyl, and otheramino acid side chains. Selection will be dependent on the availabilityof any functional group on the receptor to which the drug molecule willbe linked. The irreversible binding conjugation agent of the modifiedligand must be specific to that functional group.

2. Hydrophobicity and hydrophilicity of the conjugation agent. Areceptor in a hydrophobic environment may require a hydrophobic ligandto reach the receptor. For example, a modified ligand having ahydrophilic conjugation agent may not be able to access a correspondingfunctional group situated deep inside the hydrophobic core of thereceptor.

3. Cleavability of the conjugation agent. It may be desirable in somecases to separate a modified ligand bound to a receptor. For example, ifa toxic drug (corresponding to a parent ligand) is to be modified, asafety mechanism must be installed to preempt situations likeover-dosing. In this case, the use of cleavable conjugation agents willenable the conjugation to be reversed if complication arises. A numberof cleavable bonds may be employed for this purpose. These includedisulfide bonds, amidine, mercurial group, vicinal glycol, azo, sulfone,ester and thioester linkages. In this regard, the conjugation agentitself may be cleavable or, if the conjugation agent is attached to themodified ligand through a spacer, the spacer may be cleavable. Thespacer in this regard can be selected from the group consisting of(N-succinimidyl 3-(2-pyridyldithio)propionate,succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene,3-(2-pyridyldithio)propionyl hydrazide, disuccinimidyl tararate,N-[4-(azidophenylazo)-benzyol]-3-aminohexyl-N′-oxysuccinimide ester,4-4′-difluoro-3,3′-dinitrophenyl-sulfone,3-(4-azido-2-nitrobenzoylseleno)propionic acid, 2-methylmaleicanhydride.

4. Size and geometry of the conjugation agent. Presence of theconjugation agent on a modified ligand must permit binding of thatligand to the receptor. Upon binding of the modified ligand to thereceptor, the conjugation agent must be in close proximity to thereceptor moiety so that conjugation is effected. A spacer as hereindefined may be required to bring the conjugation agent in closeproximity to the receptor moiety. In such a case, the length of thespacer will be dependent on the distance required for the conjugationagent to reach a conjugation-effective location adjacent to the receptormoiety. Structural analysis of receptor using X-ray crystallography,nuclear magnetic resonance and the like, combined with molecularmodeling, can be of assistance in identifying the receptor moiety to beconjugated and in selecting and configuring the conjugation agent on theligand chemical structure. The receptor moiety may be present in theligand-binding site of the receptor. Preferably, the receptor moiety ispresent outside the ligand-binding site of the receptor. While not beinglimited to any theory, it is possible that this latter approach may beadvantageous since it would be more likely to prevent disruption ofreceptor function upon binding with the modified ligand. On this basis,a parent agonist may be modified such that it crosslinks with itscognate receptor to thereby cause continuous stimulation of receptorfunction (“receptor turn-on”). Alternatively, a parent antagonist may bemodified such that it crosslinks with its cognate receptor to therebycause continuous inhibition of the receptor function (“receptorturn-off”).

3. Compositions

The invention also encompasses a composition comprising the modifiedligand as described herein, together with a pharmaceutically acceptablecarrier.

The invention also features a method of treatment or prophylaxis of acondition associated with a target receptor, comprising administering toa patient in need of such treatment a therapeutically effective dosageof the composition as broadly described above.

Depending upon the particular route of adinistration, a variety ofpharmaceutically acceptable carriers, well known in the art may be used.These carriers may be selected fiom sugars, starches, cellulose and itsderivatives, malt, gelatine, talc, calcium sulphate, vegetable oils,synthetic oils, polyols, alginic acid, phosphate buffered solutions,emulsifiers, isotonic saline, and pyrogen-free water.

Any suitable route of administration may be employed for providing apatient with a composition of the invention. For example, oral, rectal,parenteral, sublingual, buccal, intravenous, intra-articular,intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular,intraperitoneal, intracerebroventricular, transdermal and the like maybe employed.

Dosage forms include tablets, dispersions, suspensions, injections,solutions, syrups, troches, capsules, suppositories, aerosols,trnnsdermal patches and the like. These dosage forms may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of a modified ligandmay be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, controlled release may beeffected by using other polymer matrices, liposomes and/or microspheres.

Compositions suitable for oral or parenteral administration may bepresented as discrete units such as capsules, sachets or tablets eachcontaining a pre-determined amount of one or more immunogenic agents ofthe invention, as a powder or granules or as a solution or a suspensionin an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion ora water-in-oil liquid emulsion. Such compositions may be prepared by anyof the methods of pharmacy but all methods include the step of bringinginto association one or more modified ligands as described above withthe carrier which constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the modified ligands of the invention with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product into the desired presentation.

The modified ligands may be in the form of a pharmaceutically acceptablesalt as is known in the art.

The above compositions may be administered in a manner compatible withthe dosage formulation, and in such amount as is therapeuticallyeffective. In this regard, the dose of modified ligand administered to apatient should be sufficient to effect a beneficial response in thepatient over time such as an amelioration of the condition to betreated. The quantity of the modified ligand(s) to be administered maydepend on the subject to be treated inclusive of the age, sex, weightand general health condition thereof. In this regard, precise amounts ofthe modified ligand(s) for administration will depend on the judgementof the practitioner. In determining the effective amount of the modifiedligand to be administered in the treatment or prophylaxis of thecondition associated with the target receptor, the physician mayevaluate progression of the condition.

In any event, those of skill in the art may readily determine suitabledosages of the modified ligands of the invention. Such dosages may be inthe order of nanograms to milligrams of the modified ligands of theinvention.

4. Detection of Target Receptors and Cells or Cell Membranes ContainingSame

The invention also features a method of detecting the presence of atarget receptor in a test sample. The method comprises contacting thesample with a modified ligand as described in Section 2, wherein saidmodified ligand binds the target receptor, and detecting the presence ofa complex comprising said modified ligand and said receptor in thecontacted sample.

The invention also encompasses a method of quantifying the presence of atarget receptor in a test sample. The method comprises contacting thesample with a modified ligand as broadly described above, wherein themodified ligand binds said target receptor, measuring the concentrationof a complex comprising the modified ligand and the receptor in thecontacted sample, and relating the measured complex concentration to theconcentration of the receptor in the sample.

The invention also provides a method of detecting the presence of atarget receptor on a cell or cell membrane. The method comprisescontacting a sample containing the cell or cell membrane with a modifiedligand as broadly described above, wherein the modified ligand binds thetarget receptor, and detecting the presence of a complex comprising themodified ligand and the cell or cell membrane in the contacted sample.

Also encompassed by the invention is a method of quantifying thepresence of a target receptor on a cell or cell membrane. The methodcomprises contacting a sample containing the cell or cell membrane witha modified ligand as broadly described above, wherein the modifiedligand binds said target receptor. The concentration of a complexcomprising the modified ligand and the cell or cell membrane is thenmeasured in the contacted sample, and the measured complex concentrationis related to the concentration of the receptor present on the cell orcell membrane.

The modified ligands can be used as a tool to identify the actual drugpocket or drug binding area on the receptor molecules. Since the bindingof these modified ligands to their targets is irreversible, the actualsite that the drugs interact with can be identified on the receptorsusing various techniques such as peptide finger printing using MassSpec. This information can be used to identify molecules that bind tothat particular drug pocket.

Any suitable technique for determining formation of the complex may beused. For example, a modified ligand according to the invention, havinga reporter molecule associated therewith (sometimes referred to hereinas a “probe”) may be utilised in any suitable assay known in the art fordetecting and/or quantifying ligand-receptor interactions. For example,scintillation counting, autoradiography, fluorography, flow cytometry,UV spectroscopy, fluorescence spectroscopy, chemiluminescence imaging,fluorescence microscopy, confocal microscopy, electron microscopy, etcmay be used in this regard.

The reporter molecule may be associated with the any suitable part ofthe modified ligand including the conjugation agent and the spacer, ifincluded. Preferably association of the reporter molecule with themodified ligand is selected such that the reporter molecule does notinterfere with binding of the modified ligand to the receptor.

It will be appreciated that the reporter molecule associated with theantigen-binding molecule may include the following:

direct attachment of the reporter molecule to the modified ligand;

indirect attachment of the reporter molecule to the modified ligand;i.e., attachment of the reporter molecule to another assay reagent whichsubsequently binds to the modified ligand; and

attachment to a subsequent reaction product of the modified ligand.

The reporter molecule may be selected from a group including achromogen, a chromophore, a catalyst, an enzyme, a fluorochrome, achemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), aradioisotope, a spin label, and a direct visual label.

In the case of a direct visual label, use may be made of a colloidalmetallic or non-metallic particle, a dye particle, an enzyme or asubstrate, an organic polymer, a latex particle, a liposome, or othervesicle containing a signal producing substance and the like.

A large number of enzymes suitable for use as reporter molecules isdisclosed in U.S. Pat. No. 4,366,241, U.S. Pat. No. 4,843,000, and U.S.Pat. No. 4,849,338. Suitable enzymes usefuil in the present inventioninclude alkaline phosphatase, horseradish peroxidase, luciferase,β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and thelike. The enzymes may be used alone or in combination with a secondenzyme that is in solution.

Suitable fluorochromes include, but are not limited to, fluoreceinisothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC),R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromesinclude those discussed by Dower et al. (International Publication WO93/06121). Reference also may be made to the fluorochromes described inU.S. Pat. No. 5,573,909 (Singer et al), U.S. Pat. No. 5,326,692(Brinkley et at). Alternatively, reference may be made to thefluorochromes described in U.S. Pat. Nos. 5,227,487, 5,274,113,5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276,5,516,864, 5,648,270 and 5,723,218.

Preferably, the reporter molecule is a radioisotope such as ³H, ¹²⁵I,¹⁴C, ³²P, ³³P, and ³⁵S.

5. Applications

Therapeutic. The modified ligands of the invention, which preferablyrepresent irreversible binding drugs, have substantial advantages overconventional reversible binding drugs. Firstly, the dose-relatedinhibition or stimulation can be persisted long after the drugsdisappear from the plasma. In other words, the drug effect is likely tolast longer than would be predicted from its plasma eliminationhalf-life. Secondly, due to longer lasting drug effects, theseirreversible binding drugs can be administered less frequently and at alower dosage. This minimizes adverse side effect and prevent cumulativetoxicity induce by the drug themselves or by their metabolites. Thirdly,since the binding of the irreversible antagonist to its receptor ispermanent, the blockade of receptor response is not longer a competitiveinhibition mechanism. This irreversible antagonism prevents the agonist,at any concentration, from producing a maximum effect on a givenreceptor. Furthermore, if the modified ligand is rendered highlyradioactive, it may be used as a therapeutic for specifically killingcells bearing the cognate receptor or may be used for imaging.

Diagnostic. Another application of this ligand modification technologyis to further elucidate various receptor subpopulations that can betargeted to relieve dysfunctions in the various complex physiologicalprocesses. More importantly, the modified drugs allow us todevelop/identify animal models that are “deficient” in certain receptorswithout undergoing lengthy, tedious and complicated manipulation of thegenetic materials. Hence the complex physiological mechanisms andfunctions of various receptors in “real-life” situations can be studiedand analyzed. Furthermore, one can also study how the various differentreceptors are interlinked and influenced by each other's functions. Theavailability of such animal models will also enable investigators topredict and reveal therapeutic outcomes of various drugs bysimultaneously blocking multiple receptor populations of interest. Thus,the present invention can be used to profile different receptors presenton a cell as well as in a tissue, organ or system. Such receptorprofiling can be used advantageously to discover novel drug targets, topredict the possible side-effects of drugs and to determine how variouscells communicate with each other, their state of health, and whetherthey respond to certain external stimuli (e.g., to drugs).

The invention will now be described with reference to the followingnon-limiting examples.

EXAMPLES Example 1

Effects of N-ethylmaleimide on the Equilibrative Nucleoside Transporterin Murine Myeloma SP2/0-Ag14 Cells.

Exemplary N-maleimide derivatives were used as a sulfhydryl reagent thatis advantageously specific to the sulfhydryl group, reacting only withcertain accessible sulfhydryl groups on the proteins, making it possiblefor specific inhibitions, and good penetration into cells due to theuncharged nature of the compound. N-Ethylmaleimide (NEM) is the smallestmaleimide reagent capable of forming stable thio esters with thereactive sulfhydryl groups of proteins. The sensitivity of es and einucleoside transport systems towards NEM was demonstrated. Variousdifferent cell lines were used to show that the sensitivity ofnucleoside transport system(s) toward NEM is a general phenomenon andnot restricted to certain cell or tissues types.

Previous studies of the es nucleoside transporter system (Paterson etal. (1977), Mol. Pharmacol., vol. 13, pp. 114; Gati et al. (1983), Mol.Pharmacol., vol. 23, pp. 146-1520) suggested that the sugar component ofnucleosides was important for binding of a nucleoside to the esnucleoside tranporter site. Thus in the present invention, that linkermoiety was attached to the pyrimidine/purine ring of the nucleosides toavoid destruction of effective inhibition for the es nucleosidetransporter to provide novel probes for that tranporter regulatory site.

The present inventor has found that a novel group of nucleosides,cytidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylic acid (CrMCC) andderivatives and analogues thereof, have the ability to irreversibleinhibiting the es nucleoside transporter proteins of human cells. Adirect, rapid synthetic route used to synthesize CrMCC or1-[[4-[(4amino-1-β-D-ribofuranosyl-2(1H)-pyrimidione)carbonyl]cyclohexyl]methyl]-1H-pyrrole-2,5-dione,is set out in FIG. 3.

To demonstrate the sensitivity of es and ei nucleoside transport systemsto NEM, various mammalian cell lines were treated with varyingconcentrations of NEM from 0 to 30 mM for 1 min prior transport assay.FIG. 1 shows the equilibrative transport (measured at 5 s uptakeinterval) of ³H-uridine (final concentration 50 μM) in murine myelomaSP2/0-Ag14 cells was inhibited by NEM in an apparent biphasic manner.About 60-70% of the transport activity was inhibited by NEM with IC₅₀value of 0.15 mM. The remaining 30-40% of transport activity wasgradually abolished by NEM at concentrations above 3 mM. Similarbiphasic inhibition by NEM was also observed in human HL-60 and humanMCF-7 cells, which possess both the es and ei trasport systems (Lee etal. (1995), Biochim. Biophys. Acta, vol. 1268, pp. 200-208). For cellsthat possess only es (murine EL-4 cells) or only ei (rat Morris 7777)transport system, monophasic ³H-uridine transport curves in NEMdose-response experiments were observed. The IC₅₀ values wereapproximately 0.1 and 1.5 mM for EL-4 and Morris 7777 cells,respectively. These results suggested that the biphasic curve of total³H-uridine transport observed in NEM dose-response experiments is areflection of the presence of two distinct equilibrative nucleosidetransport systems in those cells. The NEM-sensitive component is the estransport system and the NEM-insensitive component is the ei transportsystem. The ei transporter, although less sensitive to NEM inhibition,can be inhibited at higher concentrations of NEM or by prolongedexposure to NEM. This observation is in agreement with the generalnotion that sulfhydryl groups of enzymes display a considerablevariation in their reactivity, ranging from unreactive, through severalstages of sluggishness, to free and being immediately reactive.

To further demonstrate that the reduction in uridine influx by estransport system is due to NEM-induced chemical modification on thecarrier protein which subsequently affected the transport affinity, andto confirm the notion that the changes in es nucleoside transportactivity can be demonstrated by changes in ³H-NBMPR binding ability,murine myeloma SP2/0-Ag14 cells pretreated with or without 0.3 mM NEMfor 1 min were assayed for the availability of high-affinity ³H-NBMPRbinding sites on the cell membranes. FIG. 2 shows that the K_(d) value(corrected for non-specific binding determined in the presence of 20 μMof NBTGR) for ³H-NBMPR binding was changed significantly in response toNEM treatment. Shortly after 1 min of NEM exposure, the apparent K_(d)value was 1.9±0.4 nM, as compared to 0.16±0.02 nM for untreated cells.However, the B_(max) values of 40,000±2,600 and 41,000±1,000 sites/cellfor NEM treated and untreated cells, respectively, were notsignificantly different. These results provide additional evidence thata critical disulfhydryl bond is located near or close to the nucleosidetransporting/binding site of the es transport system, where formation ofdisulfhydryl bond with NEM affects the transport affinity of thecarrier. The suggestion that the critical sulfhydryl group on the estransporter protein is probably located or close to but not on thenucleoside trasporting/binding site is derived from the observationsthat NBMPR, dilazep, dipyridamole at 30 μM and uridine at 10 mM wereincapable of protecting this sulfhydryl group from NEM modification (Leeet al. (1995), Biochim. Biophys. Acta, vol. 1268, pp. 200-208). AlthoughNEM is effective as an inhibitor of es transporter protein, it is toxicand should be modified for therapeutic purposes.

Example 2

Synthesis and Characterization of CrMCC

The strategy to selectively irreversibly inhibit the es transporterprotein is to attach a reactive group specific covalent binding agent(maleimide) to a driver so that it can deliver the covalent bindingagent to the desired target. The most suitable driver will be thephysiological ligand itself (nucleoside). Cytidine, a pyrimidinenucleoside, is selected among other physiological nucleosides due to its“function inertness”, thus minimizes “non-specific” drug binding. As forthe spacer arm that links the maleimide to the cytidine, a cyclohexanecarboxylic acid is chosen for pilot studies. This configuration is tomimic the chemical structure of NBMPR (FIG. 5 b). Furthermore, otheradvantages like hydrophobicity (provided by cyclohexane) and stability(provided by carboxylic acid) are also taking into consideration.

A direct, rapid synthetic route used to synthesize CrMCC or1-[[4-[(4-amino-1-β-D-ribofuranosyl-2(1H)-pyrimidione)carbonyl]cyclohexyl]methyl]-1H-pyrrole-2,5-dione, is set out in FIG. 3.

N-Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) andcytidine were dissolved in anhydrous dimethysulfoxide (DMSO) separatelyprior reaction. The reaction started when these two reagents were mixedat room temperature and shielded from light. The molar concentrationratio of SMCC:cytidine in the mixture was 0.10 M:0.15 M with pH 7.5-8.0in the reaction system. Within 4 hrs, CrMCC was found in the reactionmixture and can be separated from cytidine and SMCC by a C₁₈reversed-phase column (Resource RPC, Pharmacia) operated on a HPLC (AKTAPurifier, Pharmacia) using the absorbance wavelength of 300 nm. Cytidinewas eluted by 100% water and CrMCC and SMCC were eluted by 15%acetonitrile in water with the flow rate 1.5 ml/min. The hydrophobicityof CrMCC is greater than that of cytidine but is less than that of SMCC.Formation of CrMCC reached its near maximum after 48 hrs of reactionbetween SMCC and cytidine at 22-24° C. (FIG. 4). The area under the peakwas calculated using a computer program UNICORN (version 2.0).

HPLC purified CrMCC was lyophilized by freeze-drying. The activity ofCrMCC was stable for at least three months if it was stored at −20° C.in a desiccator. The molecular weight of CrMCC was determined by LC-MS(Waters). Briefly, CrMCC was eluted out of a C₁₈ reversed-phase column(Resource RPC, Pharmacia) by 20% acetonitrile in water at a flow-rate of3 ml/min. The capillary voltage of micromass mass spectrometer was setto 3.0-3.5 V, and the cone voltage was set at 20 V. N₂ gas flow was at700 L/hr and electrospray was negative. Mass spectra were gathered undera full-scan operation, scanning range 1-1000 m/z. The molecular weightof CrMCC was determined by monitoring the protonated molecular ion, andwas similar to the predicted value of 462.5. The purity of CrMCCsynthesized was consistently greater than 95%.

The light absorbance spectrum of CrMCC was measured by uv-visspectrophotometer. FIG. 5A shows there are two λ_(max)'s for the lightabsorbance spectrum of CrMCC. One of them is at 254 nm (similar to theλ_(max) of cytidine) and the other one is at 300 nm (similar to theλ_(max) of SMCC). Such an absorbance profile of CrMCC with two λ_(max)'sis very similar to that of NBMPR (FIG. 5B). This indicated that CrMCCconsists of both pyrimidine and cyclohexane rings on its chemicalstructure. Subsequent structural studies using NMR confirmed thisfinding.

Example 3

Effects of CrMCC on the Binding of ³H-NBMPR in Human HL-60 PromyelocyticLeukemia Plasma Membranes

To synthesize ³H-CrMCC, radioactive cytidine (³H-cytidine) was used.Since high concentration of substrate increases the yield of CrMCC,nonradioactive cytidine was pre-mixed with radioactive ³H-cytidine at aconcentration ratio of 100:1 prior to reaction with SMCC (see Example2).

Purified HL-60 plasma membranes suspended in reaction buffer (0.13 MNaCl, 0.02 M NaHCO₃, pH 7.0) were pretreated with graded concentrationsof cytidine, SMCC, and CrMCC for 5 min prior exposed to ³H-NBMPR (5 nM)for additional 30 min. The reaction was terminated by membranefiltration method (Lee & Jarvis (1988), Biochem. J., vol. 249, pp.557-564). The data shown in FIG. 6 were corrected for non-specificbinding determined in the presence of 20 μM of NBTGR. FIG. 6 shows thespecific of ³H-NBMPR to HL-60 unsealed plasma membranes was inhibited byCrMCC with IC₅₀ value of 10 μM. In contrast, the IC₅₀ values for SMCCand cytidine in inhibiting ³H-NBMPR binding were 100 μM and >1 mM,respectively. The K_(i) value for CrMCC was calculated to be 1 μM.

To analyse the effect of CrMCC on the binding kinetics of ³H-NBMPR,purified HL-60 plasma membranes were pretreated with 0, 10, and 50 μM ofCrMCC, for 5 min prior incubated with graded concentrations of ³H-NBMPR(0.2 to 8 nM) for additional 30 min. A double reciprocal plot of theresults is presented in FIG. 7. The lines of the plots were intersectedat the abscissa indicating a changed value for B_(max) but an unchangedvalue for K_(d) in the presence of CrMCC. For the data shown, theapparent K_(d) values of ³H-NBMPR binding were 0.36±0.04, 0.31±0.03 and0.39±0.05 nM with B_(max) values of 1.56±0.05, 0.59±0.01 and 0.30±0.01pmol/mg protein for membranes treated with 0, 10 and 50 μM of CrMCC,respectively. Data were corrected for non-specific binding determined inthe presence of 20 μM of nitrobenzylthioguanosine (NBTGR), anon-radioactive competitive ligand. These results suggested anon-competitive inhibition of ³H-NBMPR binding by CrMCC. This is aunique feature of the irreversible antagonism.

Any clinically useful drugs must have little or no cytotoxicity at theirtherapeutic dosage range. HL-60 cells in logarithmic growth at aninitial cell density of 5×10⁴ cells/ml in RPMI medium containing 10% FBSwere exposed to graded concentrations of CrMCC, cytidine, and SMCC (0 to100 μM) for 3 days. The cell density was counted using an electronicparticle analyzer (Sysmex). FIG. 8 shows both CrMCC and cytidine hadlittle or no effect on HL-60 cell growth at concentrations as high as100 μM after 3 days of exposure. In contrast, SMCC, one of the parentcompounds, was extremely toxic to HL-60 cells with IC₅₀ value of lessthan 0.5 μM on cell growth The toxicity of SMCC is attributed to itsnon-specific interaction with every accessible sulfhydryl groups on thecells. Little or no inhibition on cell growth by cytidine is expected asthis nucleoside is rather “inert” and does not induce nucleotideimbalance at concentration range tested.

Example 4

Binding of ³H-CrMCC to the Human HL-60 Promyelocytic Leukemia PlasmaMembranes

The availability of radioactive CrMCC (³H-CrMCC) makes it possible tostudy the biochemical properties of CrMCC on the es nucleosidetransporter. This experiment was conducted to investigate the rate of³H-CrMCC binding to the unsealed plasma membranes of HL-60 cells. FIG. 9shows the binding of ³H-CrMCC (30 μM final concentration) to purifiedHL-60 plasma membranes was rather slowed. A minimum of 5 min is neededto achieve the maximum binding value of 12 nmoles per mg of HL-60 plasmamembrane protein. This is unlike the binding of reversible antagonistssuch as NBMPR, dipyridamole and dilazep, which the binding was known tobe rapid and mostly completed within first minute of incubation. Thebinding reaction in FIG. 9 was terminated by membrane filtration methodand the data were corrected for filter blanks.

It is important to confirm the binding of ³H-CrMCC to the unsealed HL-60plasma membranes is indeed irreversible. Purified unsealed HL-60 plasmamembranes were incubated with 30 μM of ³H-CRMCC for 10 min. After whichthe mixtures were diluted 20 folds and the diluted mixtures were sat atroom temperature for various time intervals to allow dissociation tooccur. FIG. 10 shows little or no dissociation of ³H-CrMCC from itsbinding sites occurred for at least 60 min after a 20-fold dilution.Even 1 mM of cytidine presence in the dilution medium failed to displace3H-CrMCC from its binding sites. In contrast, the binding of 30 μMof³H-cytidine to HL-60 plasma membranes was low and dissociated rapidlyand completely upon dilution (inset of FIG. 10). This finding togetherwith the non-competitive inhibition of ³H-NBMPR binding by CrMCC (FIG.7) suggested the interaction of CrMCC to its binding sites is indeedirreversible. The dissociation reaction shown in FIG. 10 was terminatedby membrane filtration method and the data were corrected for filterblanks.

To determine the concentration dependence of ³H-CrMCC binding to itsbinding sites, purified unsealed HL-60 plasma membranes were incubatedwith graded concentration of ³H-CrMCC (3 to 800 μM) for 10 min and thereaction was terminated by membrane filtration method (FIG. 11). Thedata can be resolved into at least two components, a high affinitycomponent (K_(d)=23.8±2.2 μM) and a low affinity component (inset ofFIG. 11). The kinetic constants for the low affinity component cannot beestimated with the existing concentration range of ³H-CrMCC. It is alsopossible that this low affinity component is a non-specific ³H-CrMCCbinding site. The data shown in FIG. 11 were corrected for filterblanks.

It is important to determine the stability of ³H-CrMCC-receptorcomplexes in various pH conditions. Purified unsealed HL-60 plasmamembranes were incubated with 30 μM of ³H-CrMCC for 5 min. The reactionmixture was then diluted 20-fold with reaction buffer of various pHvalues (pH 0.85 to 7.5). The dissociation reaction was terminated bymembrane filtration method. The data were corrected for filter blanks.FIG. 12 shows ³H-CrMCC-receptor complexes were very stable atphysiological pH. However, the ligand-receptor complexes began to breakdown at pH below 3.5.

Early studies with sulfhydryl reagent NEM had suggested that a cysteineresidue is probably situated very close to but not on the nucleosidetransporting/binding site of the es nucleoside transporter protein.Thus, if ³ H-CrMCC is bind to the same site that NEM binds, then thesubstrates that failed to inhibit NEM action (Lee et al. (1995),Biochim. Biophys. Acta, vol. 1268, pp. 200-208) should. similarly havelittle or no effect on 3H-CrMCC binding. FIG. 13 shows 1 mM ofphysiological nucleosides (e.g. cytidine, uridine, adenosine andinosine), and 20 μM of es nucleoside transport inhibitors (e.g. NBMPRand dipyridamole) indeed failed to inhibit the binding of ³H-CRMCC (30μM final concentration) to the HL-60 plasma membranes. In contrast, 1 mMof NEM was as effective as 0.5 mM of CrMCC in inhibiting the binding of³H-CrMCC. This finding suggested that NEM and CrMCC reacted to the samesulfhydryl group on the es nucleoside transporter protein.

Example 5

Effects of Other Sulfhydryl Reactive Covalent Arms on the Binding of³H-cytidine Molecule to Human HL-60 Promyelocytic Leukemia PlasmaMembranes

It was successfully demonstrated that maleimide, a sulfhydryl groupreactive agent, linked to cytidine via a cyclohexane carboxylic acidspacer arm is effective in irreversibly inhibiting the binding of³H-NBMPR to the es transporter protein. In this experiment, the bindingcapability of ³H-cytidine molecule to the HL-60 plasma membranes afterbeing linked to the same or different sulfhydryl group reactive agentvia different spacer arms was compared, to provide an indirectindication on the suitability of the spacer arms used in creating theirreversible es nucleoside transport inhibitor. Thus, ³H-cytidine waschemically modified to incorporate various sulfhydryl reactive side armsonto the 6-amino position of the pyrimidine ring. Chemicals used tomodify ³H-cytidine include m-maleimidobenzoyl-N-hydroxysuccinimide(MBS), N-succinimidyl 4-[p-maleimidophenyl]butyrate (SMPB),N-[γ-maleimidobutyryloxy]succinimide,4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridylthio]toluene (SMPI),N-succinimidyl 3-[2-pyridyldithio]propionate (SPDP), N-succinimidylmaleimidoacetate (AMAS), and N-[ε-maleimidocaproyloxy]succimide (EMCS),in addition to N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Purified HL-60plasma membranes were incubated with 100 μM of these chemically modified³H-cytidine analogs for 5 min. The reaction was terminated by membranefiltration method. FIG. 14 shows N-α-maleimidoacetoxylic acid (AMA)covalent arm promotes highest irreversible binding of ³H-cytidine to theHL-60 plasma membranes followed by 3-[2-pyridyldithio]propionic acid(PDP). However, N-ε-maleimidocaproylic acid (EMC), m-maleimidobenzoicacid (MB), N-γ-maleimidobutyrylic acid (GMB) and4-[p-maleimidophenyl]butyrylic acid (MPB) were equally effective ascovalent arms as compare to N-maleimidomethyl cyclohexane carboxylicacid (MCC). In contrast, α-methyl-α-[2-pyridyldithio]toluene carbonicacid (MPT) was least effective. Symbol X in FIG. 14 represents cytidine.

Example 6

Identification of ³H-CrMCC Binding Proteins in Human HL-60 PromyelocyticLeukemia Plasma Membranes

Attempts were made to identify the plasma membrane proteins that werelabelled by ³H-CrMCC. ³H-CrMCC (20 μM) labelled HL-60 plasma membraneproteins solubilized in SDS were loaded onto a C₁₈ reversed-phase column(Resource RPC, Pharmacia) operated on a FPLC (ATKA FPLC, Pharmacia) withthe following conditions: column volume (CV, 3 ml), starting buffer A(0.05% TFA in water), eluent B (0.065% TFA in acetonitrile), flow rate(2 ml/min), detection (280 nm), elution (0% B in 3 CV, 0-5% B in 1 CV,5% B in 5 CV, 5-100% B in 15 CV, wash-out 100% B). FIG. 15. shows thegeneral UV (λ=280 nm) absorbance profile of HL-60 plasma membranes afterbeing separated by FPLC using a C₁₈ reversed-phase column. The arrows onthe cbromatogram indicate the protein peaks that were labelled by³H-CRMCC (refer to FIG. 16). The eluents were collected at 1 ml/min andthe radioactivity was determined by a liquid scintillation counter. FIG.16 shows there are at least three major radioactive peaks located atfraction numbers 43-44, 49-50, and 57-59 of the chromatogram. The sharpradioactive peak at fraction number 6 is due to degradation product of³H-CRMCC (i.e. ³H-cytidine) and the non-specific binding of ³H-MCC tothe membrane lipids.

Example 7

Development of Irreversible Binding Drugs of Adenosine Origin

In view of the success of creating irreversible inhibitor of estransporter protein using cytidine as a lead compound, reproduction ofthe invention can be implemented by attaching a covalent arm to otherphysiological nucleosides such as adenosine (FIG. 17). ³H-Adenosine waschemically modified to incorporate various sulfhydryl reactive covalentarms onto the 6-amino position of the purine ring according to thegeneral procedure set out in FIG. 3, and using the chemicals listed inExample 5. HL-60 plasma membranes were incubated with 100 μM of thesechemically modified ³H-adenosine analogs for 5 min. The reaction wasterminated by membrane filtration method. FIG. 17 shows4-[p-maleimidophenyl]butyrylic acid (MPB) covalent arm promotes highestirreversible binding of ³H-adenosine (100 μM) to the HL-60 plasmamembranes followed by N-α-maleimidoacetic acid (AMA). However,3-[2-pyridyldithio]-propionic acid (PDP), N-ε-maleimidocaproylic acid(EMC), m-maleimidobenzoic acid (MB), and N-maleimidomethyl cyclohexanecaxboxylic acid (MCC) were equally lesser effective as covalent arms. Incontrast, N-γ-maleimidobutyrylic acid (GMB) andα-methyl-α-[2-pyridyldithio]toluene carbonic acid (MPT) were leasteffective.

Example 8

Inhibitory Effects of Irreversible Interaction 5-HT Analogs on theBinding of ³H-5-HT in Murine Brain Membranes

A. Compound Synthesis

The compounds shown in FIGS. 19 a, 20 a, 21 a and 22 a (chemicalstructures of LBT3001(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione), LBT3002(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxyl-1H-indol-3-yl)-ethyl]-butyramide),LBT3004(3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-5-hydroxyl-1H-indol-3-yl)-ethyl]-propionamide),and LBT3005 (4-(2,5-dioxo-2,5dihydro-pyrrol-1-yl-methyl)-cyclohexanecarboxylic acid [2-5-hydroxy-1H-indol-3-yl)-ethyl]-amide), respectively)demonstrated efficacy as irreversible binding compounds, and provideexamples of modified ligands which are modified 5-HT receptor bindingligands comprising conjugation agents.

The reaction scheme for synthesis of LBT3001 (FIG. 19 b) is as follows:5-Hydroxytryptamine (212.7 mg, 1.0 mmol) was dissolved in saturatedsodium bicarbonate (25 ml) solution at 0° C. N-Ethoxycarbonyleimide(177.6 mg, 1.05 mmol) was added under stirring. After 30 minutes, theice-water bath was replaced with warm water bath (30° C. to 40° C.). Thereaction solution was then stirred in the warm water bath for about onehour. The solution was then extracted with ethyl acetate (3×50 ml). Theethyl acetate layer was then washed with deionized water until pH closeto neutral (2×20 ml) and then with brine (20 ml). The organic layer wasthen dried with anhydrous MgSO₄. The solvent was removed under reducedpressure to obtain a crude product in yellow oil. Purification throughflash chromatography (30% ethyl acetate in hexane) afforded the productas a yellow to orange crystal (187.2 mg, 73%).

The reaction scheme for synthesis of LBT3002 (FIG. 20 b) is as follows:5-Hydroxytryptamine hydrochloride (42.5 mg, 0.2 mmol) and3-maleimidobutyric acid (40.3 mg, 0.22 mmol) were suspended in 1 ml2-methoxyethyl ether (DME). To the solution, N-methyl morpholine (NMM,25 μl, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22mmol) were added. The solution was stirred for 3 hours at tne roomtemperature. THe product was purified by flash chromatography directlyusing a gradient solvent eluent (0% to 3% methanol in methylenechloride) to generate brownish oil (40.7 mg, 60%).

The reaction scheme for synthesis of LBT3004 (FIG. 21 b) is as follow:5-Hydroxytryptamine hydrochloride (42.5 mg, 0.2 mmol) and3-maleimidopropionic acid (37.2 mg, 0.22 mmol) were suspended in 1 mlmethoxyethyl ether (DME). To the solution, N-methyl morpholine (NMM, 25μl, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22mmol) were added. The solution was stirred for 3 hours at the roomtemperature. The product was purified by flash chromatography directlyusing a gradient solvent eluent (0% to 1.5% to 5% methanol in methylenechloride) to generate an orange crystal (46.0 mg, 70%). The reactionscheme for synthesis of LBT3005 (FIG. 22 b) is as follow:4-Aminomethyl-cyclohexanecarboxylic acid (2.83 g, 18.0 mmol) insaturated NaHCO₃ (80 ml) was stired vigorously at 0° C. To the solution,N-ethoxycarbonyleimide (finely grounded, 3.05 g, 18.0 mmol) was addedportion by portion. When the addition was completed (c.a 10 minutes),deionized water (80 ml) was added and the mixture was sired at roomtemperature for 40 minutes. The resulting mixture was bought to pH 1-2with 1 M of HCl and extracted with ethyl acetate (3×80 ml). The organiclayer was washed with deionized water (50 ml) and then brine (20 ml).The organic phase was dried over magnesium sulfate and evaporated underreduced pressure to generate a crude product. Purification by flashchromatography (hexane:ethyl acetate:acetic acid, 60:39:1) to afford theproduct as a solid (2.69 g, 63%). 5-Hydroxytryptamine hydrochloride(42.5 mg, 0.2 mmol) and maleimidomethylcyclohexane carboxylic acid (52.2mg, 0.22 mmol) were suspended in 1 ml methoxyethyl ether (DME). To thesolution, N-methyl morpholine (NMM 25 μl, 0.22 mmol) and1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22 mmol) were added. Thesolution was stirred for 3 hours at the room temperature. The productwas purified by flash chromatography directly using a gradient solventeluent (ethyl acetate:hexane (1:1) to ethyl acetate:hexane (3:1)) togenerate a light brown oil (28.3 mg, 35.8%).

Other 5-HT receptor binding compounds such as those shown in FIG. 23 canbe similarly modified for irreversible binding to their target sitesusing one of the reaction schemes illustrated in FIGS. 19 b to 22 b.

B. Assay

FIG. 18 shows the inhibitory effects of various 5-HT analogs on the highaffiity binding of ³H-5-HT to murine brain membranes. Purified murinebrain membranes suspended in reaction buffer (0.13 M NaCl, 0.02 MNaHCO₃, pH 7.0) were pretreated with graded concentrations of LBT3001(▪), LBT3002 (□), LBT3004 (∘), and LBT3005 (●) for 5 min prior exposureto ³H-5-HT (5 nM final concentration) for additional 30 min. Thereaction was terminated by membrane vacuum filtration method. The datashown were corrected for non-specific binding determined in the presenceof 1 mM of non-radioactive 5-HT. The results were plotted agalnstcontrol binding determined in the absence of inhibitors.

FIG. 18 shows the binding of 3H-5-HT to murine brain membranes wasinhibited by all analogs of 5-HT in an apparent biphasic manner.

LBT3001 (1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-pyrrole-2,5-dione), ananalog containing no spacer arm between the 5-HT and the maleimidemolecules (FIG. 19 a), effectively inhibited the binding of ³H-5-HT toits high and low affinity binding sites with IC₅₀ values of about 0.001and 50 μM, respectively. LBT3002, an analog containing 3 carbonmolecules in the spacer arm (FIG. 20 a), inhibited the binding of³H-5-HT to its high and low affinity binding sites with IC₅₀ values ofabout 0.00003 and 200 μM, respectively. LBT3004, an analog containing 2carbon molecules in the spacer arm (FIG. 21 a), inhibited the binding of³H-5-HT to its high and low affinity binding sites with IC₅₀ values ofabout 0.2 and 50 μM, respectively. LBT3005, an analog containingcyclohexane carboxylic molecule in the spacer arm (FIG. 22 a), inhibitedthe binding of ³H-5-HT to its high and low affinity binding sites withIC₅₀ values of 0.5 and 300 μM, respectively. These results clearlyindicate irreversible binding drugs can be designed using the technologydescribed. Additionally, the technology is applicable to many smallmolecules and can be applied to further improve the efficacy of existingmolecules.

The entire disclosures of all publications, patents and patentapplications referred to herein are hereby incorporated by reference.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill appreciate that, in light of the instant disclosure, variousmodifications and changes can be made in the particular embodimentsexemplified without departing from the scope of the present invention.All such modifications and changes are intended to be included withinthe scope of the appendant claims.

1. A process for modifying a parent ligand, comprising attaching to saidparent ligand a conjugation agent that is reactive with a moiety of atarget receptor to which said parent ligand binds, wherein when saidparent ligand binds to the receptor a covalent bond is formed betweensaid conjugation agent and said moiety, and wherein the parent ligandbinds specifically with a nucleoside transporter.
 2. The process ofclaim 1, wherein the conjugation agent is attached to the parent ligandthrough a spacer.
 3. The process of claim 2, wherein the spacercomprises a group selected from the group consisting of alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl, heterooxoalkyl,alkenyl, hetero alkenyl, aralkyl, hetero aralkyl, aryl and heteroarylradicals.
 4. The process of claim 1, wherein the conjugation agent isselected from the group consisting of a sulfhydryl group specificconjugation agent, an amino group specific conjugation agent, a carboxylgroup specific conjugation agent, a tyrosine specific conjugation agent,an arginine specific conjugation agent, a histidine specific conjugationagent, a methionine specific conjugation agent, a tryptophan specificconjugation agent, and a serine specific conjugation agent.
 5. Theprocess of claim 1, wherein the conjugation agent is a sulfhydryl groupspecific conjugation agent selected from the group consisting ofN-maleimide and N-maleimide derivatives.
 6. The process of claim 5,wherein the N-maleimide derivatives are selected from the groupconsisting of disulfide reagents including 5′-dithiobis-2-nitrobenzoicacid), 4,4′-dithiodipyridine, methyl-3-nitro-2-pyridyl disulfide, andmethyl-2-pyridyl disulfide.
 7. The process of claim 1, wherein theconjugation agent is selected from the group consisting of alkylatingagents and acylating agents.
 8. A process for modifying a parent ligand,comprising attaching to said parent ligand a sulfhydryl group specificconjugation agent that is reactive with a sulfhydryl group of a targetreceptor to which said parent ligand binds, wherein when said parentligand binds to the receptor a covalent bond is formed between saidconjugation agent and said sulfhydryl group, and wherein the parentligand binds specifically with a serotonin receptor.
 9. A modifiedligand produced by the process of claim
 1. 10. A modified ligandproduced by the process of claim
 8. 11. A modified ligand having thegeneral formula:L-R₁-A   (I) wherein L is a parent ligand that binds specifically with atarget receptor comprising a nucleoside transporter; wherein A is aconjugation agent that is reactive with a moiety of the target receptorto which the parent ligand binds, such that when said parent ligandbinds to the receptor a covalent bond is formed between said conjugationagent and said moiety; and R₁ is an optional spacer.
 12. The modifiedligand of claim 11, wherein the spacer comprises a non-hydrolysableradical selected from the group consisting of alkyl, heteroalkyl,cycloalkyl heterocycloalkyl, oxoalkyl, heterooxoalkyl, alkenyl, heteroalkenyl, aralkyl, hetero aralkyl aryl and heteroaryl radicals.
 13. Themodified ligand of claim 11, wherein the conjugation agent is asulthydryl group specific conjugation agent.
 14. The modified ligand ofclaim 12, wherein the sulfhydryl group specific conjugation agent isselected from the group consisting of N-maleimide and N-maleimidederivatives.
 15. The modified ligand of claim 11, wherein the parentligand binds specifically with an es nucleoside tporter.
 16. Themodified ligand of claim 11, which is interactive with es nucleosidetransporter and/or nucleoside/nucleotide/nucleobase-sensitive proteins,wherein said modified ligand has a general formula selected from thegroup consisting of:

wherein A is N-maleimide, 2-pyridyldithio, or halogen; X is NH, S, or O;Y is H, halogen, NH₂, or O; Z is H, halogen, or CH₃; R₁ is spacer arm;and R₂ is H, β-D-ribose, β-D-2-deoxyribose, or their 5′-mono-, 5′di-,and 5′tri-phosphate.
 17. The modified ligand of claim 11, which has ageneral formula selected from the group consisting of:

wherein R₄ is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], orN-[ε-caproylate];

wherein R₄ is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], or N-[ε-caprylate];

wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or6-[3′-propioarido]hexanoate; and

wherein R₅ is 4-carbonyl-α-methyl-α-toluene,6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or6-[3′-propioamido]hexanoate.
 18. A modified litany having the generalformula:L-R₁-A   (I) wherein L is a parent ligand that binds specifically with atarget serotonin receptor; wherein A is a conjugation agent that isreactive with a sulfhydryl group of said target receptor to which theparent ligand binds, such that when said parent ligand binds to thereceptor a covalent bond is formed between said conjugation agent andsaid sulfhydryl group; and R₁ is an optional spacer.
 19. The modifiedligand of claim 18, wherein the parent ligand comprises serotonin or aprecursor or analog thereof.
 20. The modified ligand of claim 18,wherein the modified ligand has a structure selected from the groupconsisting of the structures shown below:

LBT3001 (1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione)

LBT3002(4-2,5-dioxo-2,5-dihydro-pyrrol-1-yl)N-[2-5-hydroxy-1H-indol-3-yl)-ethyl]-butyramide)

LBT3004 (3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-propionamide); and

LBT3005 (4-(2,5dioxo-2,5dihydro-pyrrol-1-ylmethyl)-cyclohexanecarboxylic acid [2-5-hydroxy-1H-indol-3-yl)-ethyl]-amide)
 21. Acomposition comprising the modified ligand of claim 11 and apharmaceutically acceptable carrier.
 22. A composition comprisingthe-modified ligand of claim 18 and a pharmaceutically acceptablecarrier.
 23. A method of detecting the presence of a target receptor ina test sample, comprising: contacting said sample with the modifiedligand of claim 11, wherein said modified ligand binds said targetreceptor if present in said test sample; and detecting the presence of acomplex comprising said modified ligand and said receptor in saidcontacted sample.
 24. A method of quantifying the presence of a targetreceptor in a test sample, comprising: contacting said sample with themodified ligand of claim 11, wherein said modified ligand binds saidtarget receptor if present in said test sample; measuring theconcentration of a complex comprising said modified ligand and saidreceptor in said contacted sample; and relating said measured complexconcentration to the concentration of said receptor in said sample. 25.A method of detecting the presence of a target receptor in a testsample, comprising: contacting said sample with the modified ligand ofclaim 18, wherein said modified ligand binds said target receptor ifpresent in said test sample; and detecting the presence of a complexcomprising said modified ligand and said receptor in said contactedsample.
 26. A method of quantifying the presence of a target receptor ina test sample, comprising: contacting said sample with the modifiedligand of claim 18, wherein said modified ligand binds said targetreceptor if present in said test sample; measuring the concentration ofa complex comprising said modified ligand and said receptor in saidcontacted sample; and relating said measured complex concentration tothe concentration of said receptor in said sample.
 27. A probe thatcovalently binds to a target receptor, said probe comprising themodified ligand of claim 11 having a reporter molecule associatedtherewith.
 28. Use of the modified ligand of claim 11 or of the probe ofclaim 27 in the study of target receptor function.
 29. A probe thatcovalently binds to a target receptor, said probe comprising themodified ligand of claim 18 having a reporter molecule associatedtherewith.
 30. Use of the modified ligand of claim 18 or of the probe ofclaim 29 in the study of target receptor function.